Project Summary/Abstract Human cytomegalovirus (HCMV) causes life-threatening diseases in patients with poor immune system function and long-lasting neurological disabilities in congenitally-infected newborns. In addition to its medical importance, HCMV and related viruses provide a powerful experimental model for investigating how viruses evolve to elude host cell defense systems. The long-term goal of this research is to discover evolutionary and molecular mechanisms by which viruses like HCMV control the host cell's protein synthesis machinery to allow viral replication. One of the defense systems that inhibits many viruses is mediated by protein kinase R (PKR). HCMV encodes two protein antagonists of PKR, TRS1 and IRS1, at least one of which is absolutely essential for the virus to replicate in human cells. Aim 1 seeks to understand why HCMV, like several other large DNA viruses, has two genes that serve the same function. Studies of mutant viruses that have only one of the genes will be used to determine why viruses lacking TRS1 have a replication defect not observed with viruses lacking just IRS1. Other experiments will reveal whether the combination of both genes confers a benefit to the virus under conditions that more closely model natural infections than previous studies, such as when clinical strains of HCMV infect different types of cells, and when they encounter an inflammatory environment. Aim 2 will elucidate mechanisms by which CMV can adapt to overcome PKR using gene duplication and horizontal gene transfer, two processes that have had major impacts on the evolution of large DNA viruses. Aim 3 will dissect how a domain in PKR that is critical for its activity has adapted to maintain its function in defending the host, while changing enough to evade viral factors. Comparisons of variants of this domain in humans, Old World monkeys, and New World monkeys, using the powerful technique of ?deep mutational scanning? in which each amino acid in a protein is mutated to all of the other 19 alternatives, will reveal how this region of PKR has evolved and how in turn CMVs have evolved to adapt to the changes. These studies will reveal insights into the mechanisms and evolution of both viral and host factors that are in conflict and that determine whether the virus replicates or is successfully blocked by host defenses.